This invention relates to novel compositions or formulations of biological or cellular materials, including but not limited to compositions containing materials such as fungi, watermolds (Oomycetes), algae, yeasts, some bacteria, and plant, insect and animal cells, that are in the form of water-in-oil emulsions (also known as “invert emulsions”). The compositions of this invention demonstrate improved storage stability over known invert emulsions, with the material retaining viability after storage, even without refrigeration.
Materials useful in the compositions include biopesticides such as biofungicides, bioherbicides and bioinsecticides, microbial materials useful in the food, pharmaceutical, waste treatment, pulp and paper, and other industries, and, in general, plant, insect and animal cellular material. The emulsion maintains cell viability, i.e., after storage in the emulsion the stored cells can be added to media and immediately start to grow.
In more specific aspects this invention relates to novel formulations and methods of use of fungal agents such as Fusarium lateritium to control infections of plants (notably grapevines), such as Eutypa lata infections, through wounds made in cutting or pruning, and to novel formulations of entomopathogenic (insecticidal) bioactive agents such as Lagenidium giganteum and their use, for instance to control mosquitoes.
One of the most significant pathogens of the grapevine industry (whether the grapes are grown as table grapes, for raisins, or for wines) is the fungal pathogen Eutypa lata, the causative agent for Eutypa dieback, commonly known as “dying arm”. This pathogen also affects other perennial crops. The disease is spread by the release of ascospores during rainfall; they enter the vascular tissue of a vine through pruning wounds, which are susceptible to infection for up to four weeks after pruning. Infections lead to yield reduction and eventual death of the vines. However, symptoms only appear after the canker is well established, which can be 2-4 years after the initial infection. When the disease is established, incidence can range upwards of 90% in 20-year-old vineyards.
E. lata infection is typically controlled by manual application of chemical or biological fungicides to pruning wounds during susceptible periods. However, this is a very labor-intensive process as the fungicide must be manually applied (“painted on”) to the individual pruning wounds. Chemical fungicides typically remain viable for only about one week, so that 4-6 applications are needed each season. One fungicide that has been used for this purpose is benomyl. However, the primary manufacturer (DuPont) has withdrawn its registration of benomyl for all purposes.
Biological control has been investigated as an alternative to benomyl. Cladosporium herbarum and Fusarium lateritium have been found effective in trials. However, performance of F. lateritium had been found inconsistent in previous studies. For that reason, a combination of benomyl-resistant F. lateritium and benomyl was proposed in published U.S. patent application 2002/159980 (Block and VanderGheynst). The two members of this combination could be applied separately, or as a combined formulation, namely a solution containing benomyl in which the Fusarium cells are also suspended. However, this is no longer a viable option due to the withdrawal of benomyl from the market. An improved F. lateritium product would be very desirable as a benomyl replacement. However, to be commercially viable, such a product must have a reasonable storage life, i.e., it can be packaged, shipped and stored, while maintaining activity, for a period of time that can extend for months. In addition, F. lateritium must be applied directly to the pruning wound shortly after pruning. As mentioned earlier, this is typically done by manual “painting” of each wound.
Lagenidium giganteum is a watermold that parasitizes the larval stage of mosquitoes. It often is included in lists of fungi but nevertheless is more closely related to diatoms and brown algae. For purposes of convenience, however, the terms “fingi”, “fingal”, and the like, as used herein, are deemed to include L. giganteum. The life cycle begins with a motile zoospore (asexual stage) that seeks out mosquito larvae, attaching to and penetrating the cuticle via a germ tube. The fungus grows inward, eventually filling the body cavity and killing the mosquito larva. The fungus can then be released from the infected cadaver, generating more zoospores that can infect other larvae. The sexual cycle produces oospores that can maintain the fungus during unfavorable conditions, such as long periods of drought. Upon flooding, oospores release infective zoospores to start the cycle again.
A commercial product containing L. giganteum was sold for several years under the trademark Laginex® AS. This product, which no longer is on the market, was an aqueous suspension containing 40% wt./wt. L. giganteum mycelium colony forming units (CFU), a concentration measured by cell counts per liter. It was sold as a suspension for aerial spraying. One drawback of this product was the lack of long-term storage stability, a general problem with L. giganteum (see, e.g., Scholte et al., J Insect Sci. 4: 1 (2004). Additionally, the product needed to be refrigerated to maintain stability. In our work, we found that cell suspensions in aqueous systems tended towards clumping, making the resulting product uneven in composition and problematic in aerial spraying
Experience has shown that to be useful in the agrochemical or pest control field, a product must have a reasonably long storage life, including under quite different storage conditions, particularly temperature. To be easily marketed and kept available for use as needed, a product may need to withstand temperatures of 100° F. or greater, and as low as freezing. Such temperatures can be encountered in shipping or storing the products, even though attempts are made to maintain storage temperatures within a more reasonable range. Biological materials such as biopesticides are coming into use more and more with the passage of time. However, such materials are generally more sensitive to temperatures than chemicals, and so the concern over storage stability of such materials is becoming greater. Additionally, biological materials tend to have shorter shelf lives than chemical materials, even under well-controlled storage conditions. However, for marketing and distribution through the normal channels, such materials may need to be kept in storage for months, whether by the distributor, the dealer, or the purchaser.
One apparent solution to problems of storage stability would be to produce the bioactive agent in dried form and, indeed, such compositions are marketed for some bioactive agents, such as B. thuringiensis. However, drying is not suitable for all organisms. Some are too sensitive to be completely dried; others show difficulties in becoming activated or reconstituted on contact with water.
In a few cases, formulation of mycopesticides (fungal materials that act as pesticides against other organisms including other fungi and weeds) has been done in the form of so-called “invert emulsions”, i.e. water-in-oil emulsions (as opposed to the much more typical oil-in-water emulsions). Such formulations are discussed, for instance, in Amsellem et al., Phytopathology 80:925 (1990), Connick et al., Weed Technology 5:442 (1991), Womack et al., Pesticide Science 37:337 (1993), Boyette et al., Weed Science 41:497 (1993), Yang et al., Phytopathology 83:953 (1993), Boyette, Weed Technology 8:526 (1994), Egley et al., Weed Science 43:312 (1995), and Womack et al., Biological Control 6:23 (1996). These works typically involved mycoherbicides contained in a water-in-oil emulsion, in which the oil often was a vegetable oil such as corn oil. A surfactant was often used, such as Myverol 18-89, a monomeric surfactant. These publications described effectiveness of the application of the pesticide in question, with the testing being done either in glasshouses or at field research stations. However, storage stability was not an essential aspect of the formulations, as they seem to have been prepared specifically for the tests in question, and so would likely not have been stored for any significant time. In addition, the invert emulsions described in such publications proved to be difficult to handle in general, and particularly difficult to spray.
In addition to use in pest control, biological materials such as fungi and the like are useful in the biotechnology, waste treatment or remediation, pharmaceutical, food, and other industries, where they may serve as catalysts, biodegradation agents, fermentation aids, and diagnostic agents among other uses. Here again, storage-stable cultures would be advantageous. Storage-stable cultures of plant, insect, and animal cells would be valuable, for more convenient preservation and storage of such materials in general.
A control agent for E. lata that can be applied directly to a pruning wound shortly after pruning, and that will have an effective life of up to 4-6 weeks, would be a very desirable product. Likewise, a product for use in controlling mosquitoes, and containing L. giganteum that that would maintain effectiveness after storage, especially if refrigeration is not required, would be very desirable.
The compositions of this invention provide such products.